Fry stocking is a primary management strategy of the U.S. fish and wildlife Service and cooperating state fishery agencies for restoring populations of fish, for example, restoring Atlantic salmon, Salmo salar, to the New England states. In order to determine the effectiveness of this strategy in achieving management goals, identification of stocked fish is critical. Therefore, it would be useful to have a technique for marking larval (nonfeeding) fish with a readily recognizable tag or mark capable of being non-lethally detected in fry, parr, smolts, and returning adult fish.
A variety of attempts have been made to mark salmonid fry by mechanical or chemical techniques. Unfortunately, none of these methods has been adequately refined or proven practical for marking large numbers of Atlantic salmon fry with a feature that can be non-lethally detected in subsequent life stages of the fish. Kaill et al. (1990) evaluated the use of half-length coded wire tags in emergent pink salmon Oncorhynchus gorbuscha and reported short-term retention rates of 93–100% and long-term retention rate estimates of 50–84%.
This technique was tested on Atlantic salmon larvae in 1994 by U.S. Fish and Wildlife biologists at the Northeast Fishery Center, Lamar, Pa., but proved to be ineffective and impractical with this species due to the small size of fry (J. W. Fletcher, NEFC, personal communication). In general, chemical marks induced in fish from immersion in dyes or stains produce short-term marks that are detectable only for days, rather than for years. Injecting dyes and stains produces more durable marks, but the fish are subjected to greater stresses during handling and marking (Muncy et al., 1990). Immersion, injection, or feeding of fluorescent chemicals, such as oxytetracycline, can produce a mark on calcified fish tissues that is delectable under ultraviolet light or through fluorometric techniques (Muncy et al., 1990). Oxytetracycline has been used to mark teleost otoliths for subsequent age validation, but the fish must be sacrificed for mark determination. Wilson et al. (1987) reported that calcein, a compound that fluoresces green under long wave ultraviolet light, produced a detectable mark on otoliths of three species of estuarine fish afer a two hour immersion in a calcein solution of 125 mg/L.
Calcein chemically binds with calcium and shows a marked increase in fluorescence when complexed with alkaline earth metals (Wallach et al., 1959). Calcein has been used as an indicator to determine the calcium content of limestone and gypsum (Diehl and Ellingboe, 1956), for determining submicrogram quantities of cadmium (Hefley and Jaselskis, 1974), as a stain for angiography (Oncel et al., 1990), and for fitting soft hydrophilic contact lenses (Refojo et al., 1972).
Prior methods for detecting fluorescent-labeled organisms required use of a microscope outfitted with the proper filters for detecting fluorescing marks in fish or other biological materials. This created the problem of transporting an expensive, delicate microscope into outdoor and harsh environments, and required that individual specimens be placed onto the microscope stage for examination. Microscope stages are not intended for examination of whole, live specimens. Even specimens which could be partially examined via microscopy were subject to excessive manipulation and focusing time to attempt evaluation for the fluorescing mark. These procedures are prohibitory for efficiently evaluating large-sized specimens or large numbers of individuals while maintaining the life of live specimens if necessary.
One conventional detector for incident light fluorescence is the AO Vertical Illuminator for Light Fluorescence microscopy, produced by Reichert Scientific Instruments, Buffalo, N.Y., which is based on microscopy. This conventional illuminator module comprises a tungsten halogen lamp or a mercury vapor lamp to provide a source of excitation energy, collector lens system and filed diaphragm to efficiently control the beam, exciter filters to transmit selected wavelengths, dichroic beamsplitters to selectively reflect and subsequently transmit desirable wavelengths, and barrier filters to bar unwanted wavelengths. The dichroic element reflects excitation energy wavelengths up to a predetermined cutoff point. Longer wavelengths, as emitted by fluorescence energy, pass through the dichroic element and are not deflected from their paths. Excitation energy, which is beamed from the lamp through the exciter filter, is reflected by the dichroic element down through the microscope objective into the specimen. Fluorochromes within a specimen, excited by the energy beam from the objective, emit visible fluorescence energy which returns up though the microscope objective, dichroic element, and barrier filters to the microscope eyepiece or camera.
Methods for monitoring using fluorescence include that shown in Rao et al., U.S. Pat. No. 5,628,310. This method is for transdermal measurements of the fluorescence lifetime of an implanted element. This method, however, requires implantation of the fluorescent device.
Krauthamer, in U.S. Pat. No. 5,239,998, discloses a method and apparatus for detecting fluorescence emitted at a remote situs, namely, tissue stained with a voltage-sensitive dye. This method and apparatus are designed to record the electrical activity of tissues stained with voltage-sensitive dyes rather than to detect marked tissues.
Harte, in U.S. Pat. No. 4,056,724, discloses a fluorometric system for detection of sample substances derived from biological fluid, or tissue targeted with fluorochromes. This apparatus requires that the sample substance be coated over a surface on a solid substrate, which means that it cannot be used to detect fluorescence in whole animals.
Thus, there is a need for batch-marking small organisms such as fish and other animals, along with a need for practical, non-lethal field detection of the mark in order to differentiate between groups of organisms, such as hatchery-reared vs wild fish. There is a need for a portable device which can withstand the rigors of outdoor field work and be used to examine samples too large for standard microscopy.